1. Field of the Invention
The present invention relates to reagents and methods for the detection of histoplasmosis. In particular, the present invention relates to nucleic acids (DNAs) relating to the M antigen gene of Histoplasma capsulatum; to vectors and host expression systems containing these nucleic acids; to nucleic acids (RNAs) which encode the M antigen of H. capsulatum; to isolated and recombinantly-produced antigens encoded by these nucleic acids; to antibodies produced against these antigens; to methods and kits for detecting histoplasmosis using these nucleic acids, antigens and antibodies; and to vaccines for the treatment or prevention of histoplasmosis.
2. Background
Histoplasmosis is a systemic fungal disease resulting from the inhalation or, less frequently, the ingestion of spores of the fungus Histoplasma capsulatum, variety capsulatum, which is worldwide in distribution. The infection often causes acute pneumonia, or disseminated reticuloendothelial hyperplasia, or an influenza-like illness with joint effusion and erythema nodosum. Reactivated infection involves the lungs, meninges, heart, peritoneum and adrenals. Clinically inapparent or mild disease can result from limited, primary site infection of H. capsulatum in the lungs, but an often life-threatening, disseminated form of histoplasmosis can occur in immunodeficient patients, particularly the elderly, and those who have acquired immunodeficiency syndrome (AIDS). It is important to properly identify H. capsulatum from other fungal species in order to determine the proper treatment for a fungal infection.
H. capsulatum is a dangerous, dimorphic, pathogenic fungus which, under different environmental conditions, may exist as either the yeast or mold phase. The organism exists as a multicellular mycelium at room temperature in rich soils, and in organic matter, in temperate environments worldwide, and proliferates as a unicellular yeast form at 37° C., and in infected host tissues. Only the yeast phase is known to survive within tissues, or within macrophages. The unicellular yeast form reproduces by budding on specialized media at 37° C. The mold form produces multicellular filamentous colonies that consist of cylindrical tubular structures called hyphae, and may contain microconidia and macroconidia which primarily grow under appropriate soil conditions, or on specialized fungal media, at 25° C. H. capsulatum occurs throughout the world, particularly in Brazil, Africa, India, Southeast Asia and the United States, but is most commonly found in soil from the fertile river valleys (Mississippi and Missouri river valleys) of the central United States.
H. capsulatum is associated with bird (particularly black bird and seagull) and bat excrement. (See, for example, Loyd et al., Histoplasma capsulatum. In Principles and Practice of Infectious Disease (3rd ed., Coordinating ed., Mandell et al., New York, (1990)); Wheat, “Diagnosis and Management of Histoplasmosis,” Eur. J. Clin. Microbiol. Infect. Dis. 8:480 (1989).) The fungus infects the soil, and the resulting infected soil is often used as a habitat by birds and/or bats.
In addition to H. capsulatum var. capsulatum, two variants of Histoplasma exist: H. capsulatum var. duboisii (African histoplasmosis) and H. capsulatum var. farciminosum (epizootic lymphangitis of horses and mules). (See, for example, Rippon, Histoplasmosis. In Medical Mycology The Pathogenic Fungi and the Pathogenic Actinomycets (3rd ed., Saunders Company, Chapter 15 (1988)).) Many strains of H. capsulatum are currently deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852. H. capsulatum strain CDC6623, deposited under accession number ATCC 26320, is discussed in Pine et al., “Procedures for the Production and Separation of H and M Antigens in Histoplasmin, and Chemical and Serological Properties of the Isolated Products,” Mycopathlogia 61:131–141 (1977). The following other strains or variants of H. capsulatum are also deposited with the ATCC: H. capsulatum (attenuated Downs strain, filamentous phase, accession number ATCC 38904), H. capsulatum (attenuated Downs strain, yeast phase, accession number ATCC 38904), H. capsulatum (filamentous phase, accession number ATCC 11407), H. capsulatum (yeast phase, accession number ATCC 11407), H capsulatum variant duboisii (filamentous phase, accession number ATCC 32281), H. capsulatum variant duboisii (yeast phase, accession number ATCC 32281), H. capsulatum variant farciminosum (filamentous phase, accession number ATCC 58332) and H. capsulatum variant farciminasum (yeast phase, accession number ATCC 58332).
The M antigen of H. capsulatum is a pluripotent glycoprotein having a molecular mass of 94 kDa, an isoelectric point of 4.7, oligosaccharide side chains, glycosidic epitopes which are N-linked to the peptide core, and protein epitopes, which have been shown to be unique to the H. capsulatum fungus. The peptide epitopes react with human antibodies, are not affected by N-deglycosylation, and trigger the proliferation of T cells. The M antigen is an immunodominant antigen of H. capsulatum, and elicits both humoral and cell-mediated immune responses. The glycopeptide bonds present in the glycoprotein are N linked. The M antigen of H. capsulatum is considered to be the immunodominant antigen of H. capsulatum because antibodies generated against the M antigen are first to arise in infection, and are more commonly present during all phases of histoplasmosis. Because the presence of this M antigen is indicative of histoplasmosis infection, the M antigen can serve as a marker for histoplasmosis infection. However, the biological identity of the M antigen has remained unknown. One report demonstrated that M protein was a catalase, based upon its ability to react with anti-catalase antibodies.
Currently, histoplasmosis is diagnosed by culture, or by the demonstration of a rise in complement-fixing antibody titers in serum. A definitive diagnosis of an H. capsulatum infection currently requires the isolation and propagation of the fungus, which is time-consuming and lacking in sensitivity, and which is dangerous for laboratory personnel, who must take extreme caution to prevent inhalation of the pathogenic fungus, so as not to become ill with a pulmonary infection. Further, only small quantities of antigens of H. capsulatum for use as biological reagents may be prepared in this manner.
Conventional laboratory identification methods used to isolate and identify H. capsulatum include the culture of a clinical specimen at room temperature on specialized fungal media. This procedure isolates the slower growing H. capsulatum colonies from possible contaminants, such as bacteria, and from faster growing saprobic fungi. This method, however, has several disadvantages. Because the growth of H. capsulatum to a visible colony normally takes from about two to four weeks, and sometimes as long as 12 weeks, this procedure is very slow. (See, for example, Rippon, Histoplasmosis. In Medical Mycology, The Pathogenic Fungi and the Pathogenic Actinomyestes, supra.; Koneman at al., Laboratory Identification of Molds, in Practical Laboratory Mycology, (3rd ed. Williams & Wilkins (1985)); and McGinnis, Histoplasma capsulatum. In Laboratory Handbook of Medical Mycology (Academic Press (1986)).) Further, additional growth is required before the characteristic colony morphology and microscopic sporulation pattern with tuberculate macroconidia may be observed. In addition, approximately 10% of cultures produce only smooth-walled macroconidia, and some cultures fail to sporulate. Moreover, many species of fungi other than H. capsulatum, such as Blastomyces dermatitidis, Chrysosporium sp., and Sepedonium sp., produce similar colony and sporulation characteristics. Thus, additional testing is usually necessary to definitively identify the organism.
One method of converting the mycelial colony of H. capsulatum to the yeast phase is performed by subculturing the organism onto highly enriched cysteine-containing media, and incubating it at 35°–37° C. However, conversion to the yeast phase is often difficult, and may require several additional subcultures at three-day intervals.
Serologic evidence is the prime diagnostic indicator of histoplasmosis. Such evidence may be obtained with several serologic tests, such as the immunodiffusion test, which detects precipitants against the species-specific H and M antigens found in histoplasmin. (See, for example, Kaufman, “Laboratory Methods for the Diagnosis and Confirmation of Systemic Mycoses,” Clin. Infect. Dis. 14:23–29 (1992), and Wheat, “Diagnosis and Management of Histoplasmosis,” supra.)
Histoplasmin, an unpurified culture supernatant obtained from the mycelial phase of H. capsulatum grown in a chemically-defined medium containing H. capsulatum M antigens is currently used to probe both humoral and cell-mediated responses in patients with histoplasmosis. It is used for the serologic diagnosis of histoplasmosis, and as a skin test antigen to demonstrate delayed hypersensitivity to infection in skin tests for histoplasmosis. The purification of histoplasmin is described by Bradley et al, “Purification, Composition, and Serological Characterization of Histoplasmin-H and M Antigens,” Infect. Immun. 9:870–880 (1974). The preparation of H and M antigens of H. capsulatum free of heterologous antigens is described by Green et al., “Preparation of h and m Antigens of Histoplasma capsulatum Free of Heterologous Antigens,” Curr. Microbiol. 12:209–216 (1985). (See also, Pine, “Histoplasma antigens: their Production, Purification and Uses,” Contrib. Microbiol. Immunol. 3:138–168 (1977).) The preparation of antisera to the M antigen is described by Green et al., “H and M Antigens of Histoplasma capsulatum: Preparation of Antisera and Location of these Antigens in Yeast-Phase Cells,” Infect. Immun. 14:826–831 (1976). General information concerning the serodiagnosis of fungal diseases is present in L. Kaufman et al., Serodiagnosis of Fungal Diseases, in Manual of Clinical Laboratory Immunology (3rd ed., American Society for Microbiology, Washington, D.C. (1988)).
Although the M antigen of H. capsulatum is useful in immunoassays for the diagnosis of histoplasmosis, purification of the M antigen from a batch culture is a laborious and low-yield process. The use of a recombinantly-produced M antigen of H. capsulatum in such immunoassays would significantly diminish the labor necessary to obtain M antigens which are pure enough to be useful in the immunoassays, and would result in high yields of the M antigen.
A need presently exists for biological reagents which can be produced and purified quickly and safely, and in large quantities, and which can be used in diagnostic assays to rapidly, easily and accurately detect a previous or current infection by H. capsulatum, and to diagnose histoplasmosis. A need also presently exists for a method of rapidly, easily and accurately detecting a previous or current infection by H. capsulatum, and to diagnose histoplasmosis. Such biological reagents and methods would allow a clinician to improve the speed and accuracy of processing large numbers of clinical samples. Such reagents and methods would also aid the clinician in patient management, eliminate unnecessary tests, improve the speed, ease and accuracy of diagnosis and prognosis, help control histoplasmosis infection and reduce the use of unnecessary medications.
Accordingly, the present invention provides the DNA nucleotide sequence of the M antigen gene of H. capsulatum, and of related nucleotide sequences, which can be used to safely and rapidly produce, by recombinant DNA techniques, large quantities of the M antigen of H. capsulatum when inserted into a vector and placed into a suitable host for protein expression. The recombinantly-produced M antigens may be quickly and safely produced in large quantities in a pure, undegraded form. The present invention also provides the RNA nucleotide sequence which encodes the M antigen of H. capsulatum, and related nucleotide sequences. Nucleic acids, and fragments thereof, within the invention can also be used as nucleic acid probes in hybridization assays, or as primers in polymerase chain reaction assays, to detect H. capsulatum in clinical samples.
The present invention also provides the deduced amino acid sequence of the H. capsulatum M antigen. Isolated and recombinant M antigens encoded by nucleic acids within the present invention can be used as biological reagents in a wide variety of tests for histoplasmosis, such as skin tests, and immunoassays to detect a previous or current H. capsulatum infection in a tissue or fluid sample obtained from a human being or animal suspected of having, or having had, histoplasmosis. For example, these antigens can be used as skin test antigens to ascertain the cell-mediated immune status of persons who have been exposed to H. capsulatum. The nucleic acids and antigens of the invention can also be used in a vaccine for the prevention or treatment of histoplasmosis.
The present invention also provides antibodies generated against the above antigens, which can be used in a wide variety of immunoassays to detect a current infection by H. capsulatum. 
The present invention further provides methods for the detection of histoplasmosis, and related kits, using nucleic acids, antigens or antibodies within the invention.
The nucleic acids, vectors, hosts, isolated and recombinantly-produced antigens, antibodies, methods of detection and kits of the present invention permit the safe, direct, rapid, efficient, and accurate detection of a previous or current infection by H. capsulatum in a patient, and a positive diagnosis of histoplasmosis.
This patent application is believed to be the first report of the nucleotide sequence of the H. capsulatum M antigen gene, the nucleotide sequence which encodes the H. capsulatum M antigen, and of the amino acid sequence of the H. capsulatum M antigen.
3. Description of the Related Art
Zancopé-Oliveira et al., “Immunochemical Analysis of the H and M Glycoproteins from Histoplasma Capsulatum,” Clin. Diagn. Lab. Immunol. Vol. 1, No. 5, 563–568 (1994), describes the use of different physicochemical methods to characterize the M and H antigens obtained from histoplasmin.
Zancopé-Oliveira et al., “Evaluation of Cation Exchange Chromatography for the Isolation of M Glycoprotein from Histoplasmin,” Journal of Medical and Veterinary Mycology 31, 29–41 (1993), describes the development of chromatography procedures to isolate the M antigen from histoplasmin, and the monitoring of the physical, chemical and serological properties of the protein.
Zancopé-Oliveira et al., “Effects of Histoplasmin M. Antigen Chemical and Enzymatic Declycosylation on Cross-Reactivity in the Enzyme-Linked Immunoelectrotransfer Blot Method,” Clinical and Diagnostic Laboratory Immunology 1, No. 4, 390–393 (1994), describes an evaluation of the enzyme-linked immunoelectrotransfer blot (EITB) method as a suitable method for detecting antibodies present in sera from patients with histoplasmosis against M antigen, and the effect of chemical and enzymatic deglysolyation of M antigen as a means of increasing diagnostic specificity. The assay described in this article was stated to demonstrate 100% sensitivity with histoplasmosis serum samples, all of which were stated to react with the H. capsulatum M antigen.
Green et al. “Preparation of h and m Antigens of Histoplasma capsulatum Free of Heterologous Antigens,” supra., describe the use of a salt gradient elution of crude histoplasmin on CM-sepharose CL6B at pH 3.0 in a one-step procedure to isolate the H, M and non-M antigens of H. capsulatum, and free them of any C antigen common to other pathogenic fungi to produce highly-purified antigens for use in immunoassays. This reference provides (Table 4 on Page 213) the gross amino acid composition (mole percent of sixteen amino acids) of the H. capsulatum M antigen, but not the amino acid sequence thereof.
Keath, “Molecular Cloning and Sequence Analysis of yps-3, a Yeast-Phase-Specific Gene in the Dimorphic Fungal Pathogen Histoplasma capsulatum,” Microbiology 140, 759–767 (1994), describes the cloning of the H. capsulatum yeast-phase-specific (yps-3) gene to clarify the mechanisms underlying pathogenesis and morphogenesis in the fungus H. capsulatum. The nucleotide sequence of the yps-3 gene, and the predicted amino acid sequence of its product, are provided.
Deepe et al., “Immunobiological Activity of Recombinant H Antigen From Histoplasma capsulatum,” Infection and Immunity, Vol. 63, No. 8, 3151–3157 (1995), describe the isolation and sequencing of the H antigen gene of H. capsulatum, and the recombinant production of the H. capsulatum H antigen in the bacterial expression vector pET 19b.
U.S. Pat. No. 5,352,579 describes nucleic acid hybridization assay probes which are stated to be specific for H. capsulatum and no other fungi, and which have the nucleotide sequence 5 CGAAGTCGAGGCTTTCAGCATG3, or the nucleotide sequence complementary thereto. A probe having the above nucleotide sequence is stated to hybridize to the 18S rRNA of H. capsulatum corresponding to bases 172–193 of Sacchromyces cerevisiae. This patent also describes the use of helper probes having the sequence 5′TATTAGCTCTAGAATTACCACGGGTATCCAAGTAGTAAGG3, or the sequence 5′CCCCGAAGGGCATTGGTTTTTTATCTAATAAATACACCCC3′.
None of the above documents teaches or suggests the DNA nucleotide sequence of the H. capsulatum M antigen gene, the RNA nucleotide sequence which encodes the H. capsulatum M antigen, the amino acid sequence of the H. capsulatum M antigen, or the production of the H. capsulatum M antigen using recombinant DNA techniques.